The long-term goal of this project is to explore the mechanisms underlying the progression of transient to persistent B Lineage Clonal Expansions (BLCE) in middle-aged mice and the progression of persistent BLCE to late-life B cell neoplasms. We hypothesize that an ordered progression of genetic alterations underlies this age-associated progression. Our first aim is to isolate antigen-specific BLCE prior to the appearance of antigen-specific serum monoclonal immunoglobulin (mlg) and antigen-specific clonal BM plasma cells after the appearance of antigen-specific mlg or diffuse large cell lymphoma (DLCL) from DNP-human serum albumin (DNP-HSA)- or hen egg lysozyme (HEL)-immunized C57/BL/6 mice. The transient or stable antigen-specific BLCE will be isolated from cryopreserved, spleen cells obtained by partial splenectomy of 4-6- and 14-18-month-old mice. BM plasma cells secreting antigen-specific mIg will be isolated from bone marrow obtained from mice sacrificed at 19-months of age with antigen-specific mIg. The antigen-specific cells will be stained with fluorescent-labeled antigen. Results obtained in immunized mice will be confirmed by isolating spontaneous, stable BLCE, bone marrow plasma cell-secreting mIg, or DLCL. Spontaneous BLCE will be isolated from cryopreserved spleen cells obtained prior to the development of mIg or DLCL by their surface or intracellular binding of fluorescent-labeled anti-clonotypic antibody. The link between antigen-induced or spontaneous BLCE and neoplastic B cells will be established by identifying signature IgH/IgL CDR3 sequences of the neoplastic cells and their stable BLCE precursors by single-cell RT-PCR. Our second aim is to develop a genetic roadmap defining the progression of BLCE into late-life B-cell neoplasms. We shall define the genetic abnormalities that accumulate during the transformation of transient to stable BLCE and stable BLCE to mIg-secreting bone marrow plasma cells and/or to DLCL. BLCE isolated from mice prior to the development of B cell neoplasms will be analyzed for the presence somatic hypermutation of proto-oncogenes by single cell RT-PCR, for chromosomal translocations and aneuploidy by FISH and for proto-oncogene expression by quantitative RT-PCR.